Method and apparatus for determination of chemical species in body fluid

ABSTRACT

Disclosed is a method and apparatus for the non-invasive determination of one or more preselected analytes in a body fluid expressed through the skin. The fluid is collected in a dermal concentration patch and concentrated by driving off a portion of the substantial water fraction under the influence of body heat. The analyte is optimally complexed with an immobilized specific binding partner and an indicium of the presence of the analyte is visually expressed. The patch may comprise a plurality of test zones for screening for a plurality of analytes. Additional positive and negative control zones are also disclosed. The patch may comprise a feature to detect tampering with the patch to produce false negative results, especially when used in screens for drugs of abuse. In addition to being useful as a drug of abuse screen, the patch is useful for diagnostic purposes.

"This application is a continuation of application Ser. No. 07/929,628,filed Aug. 13, 1992, now abandoned, which was a continuation of Ser. No.07/569,007, filed Aug. 15, 1990, now U.S. Pat. No. 5,203,327, issuedApr. 20, 1993, which was a continuation-in-part of Ser. No. 07/241,707,filed Sep. 8, 1988, now U.S. Pat. No. 4,957,108, issued Sep. 18, 1990."

BACKGROUND OF THE INVENTION

The present invention relates to diagnostic kits for determining thepresence of one or more analytes in a fluid sample. More particularly,the present invention relates to a dermal concentration patch forincreasing the concentration of an analyte expressed through the skin toa conveniently measurable level.

The determination of a patient's physiological status is frequentlyassisted by chemical analysis for the existence and/or concentration ofpredetermined chemical species in a body fluid. These tests, which aretypically conducted in the physician's office or in the hospital, may becharacterized by their collection technique as invasive, such asanalyses of blood, or non-invasive, such as analyses of urine andperspiration.

Blood is frequently analyzed for a wide variety of components, andclinical laboratories are generally equipped with instrumentation whichcan provide a highly quantitative profile of the blood's composition.However, blood collection is inherently invasive, and therefore attendedby several disadvantages. Analyses based upon collection of a sample ofblood are generally restricted to the physician's office or clinicallaboratory, which reduces convenience for ambulatory patients andgreatly increases cost. In addition, some risks associated with aninvasive procedure can range from undesirable at best to unacceptable,depending upon the condition of the patient, and the nature andnecessity of the test desired to be performed.

Many analytes or metabolites of interest can additionally be detected inurine, which is characterized by its predictable supply and non-invasivecollection. However, as will become apparent, urine analysis is not wellsuited for use in the principal intended application of theconcentration patch of the present invention.

Perspiration is, under certain circumstances, an ideal body fluid foranalysis in the determination of physiological status. Its non-invasivecollection renders it suitable for use out of the physician's office,and its similarity to blood in terms of its content of biologicalmolecules renders it suitable for a wide range of physiological testing.

Thus, a variety of diagnostic kits for monitoring an analyte in sweathave been developed. For example, U.S. Pat. No. 3,552,929 to Fields, etal. discloses a band-aid-type test patch particularly suited fordetermining the chloride ion concentration in perspiration as a methodof diagnosing cystic fibrosis. The apparatus disclosed in Fieldscomprises an absorptive sweat collecting pad with an impermeableoverlying layer for the purpose of preventing evaporation. When theabsorptive pad is saturated, the patch is removed from the skin andexposed to a series of strips impregnated with incremental quantities ofsilver chromate or silver nitrate, the color of which undergoes a wellknown change upon conversion to the chloride salt.

U.S. Pat. No. 4,706,676 to Peck discloses a dermal collection devicewhich comprises a binder to prevent reverse migration of an analyte, aliquid transfer medium which permits transfer of an analyte from thedermal surface to the binder, and an occlusive cover across the top ofthe liquid transfer medium and binder.

Peck discloses application of the dermal collection patch in thedetection of human exposure to various environmental chemicals. Afterthe dermal collection device has been worn on a patient's skin for aperiod of time, the patch is removed for analysis. Analysis involveschemical separation of the bound substance of interest from the bindingreservoir and thereafter undertaking qualitative and/or quantitativemeasurement by conventional laboratory techniques.

The prior art generally suffers from one or more important limitationswhen convenient field use of a diagnostic test patch is desired. Inparticular, prior art diagnostic test patches are generally only usefulfor determining the presence of analytes such as halide ions, which arepresent in sweat in relatively high concentrations. Other prior artdermal patches are merely collection devices from which the analytesmust later be separated and concentrated or otherwise prepared foranalysis in accordance with known laboratory techniques. In addition,the occlusive outer layer type devices of the prior art are susceptibleto the problem of back diffusion of perspiration and/or analytescontained therein.

Thus, there remains a need in many diverse applications for a method andapparatus for the non-invasive determination of a preselected analyte ina body fluid such as perspiration, which can be utilized to detect thepresence of low-concentration analytes in perspiration without the needfor conventional instrumentation. Additionally, these remains a need fora method and apparatus for the non-invasive determination of apreselected analyte in insensible or non-exercise perspiration. The testkit should be low-cost and suitable for convenient use by non-medicalpersonnel.

SUMMARY OF THE INVENTION

There is provided in accordance with one aspect of the present inventiona dermal concentration patch for concentrating components of a bodyfluid under the influence of body heat, which comprises a concentrationzone in communication with a source of body fluid and a discharge zonewhich is exposed to the atmosphere to permit escape of at least aportion of the substantial water component and other undesiredcomponents in the body fluid.

In one embodiment, a hydrophobic membrane is provided to separate theconcentration zone from the discharge zone. By hydrophobic, it is meantin the context of the present invention that the membrane prevents thepassage of fluid phase but permits the escape of vapor phase of volatilecomponents. These hydrophobic membranes shall also be referred to hereinas gas permeable membranes. When a gas permeable membrane is provided,the transition of body fluid accumulated in the concentration zone tothe vapor phase is accelerated under the influence of body heat, therebyconcentrating the non-volatile and less volatile components in theconcentration zone.

Alternatively, the concentration zone and the discharge zone may beseparated by a hydrophilic layer. By hydrophilic, it is meant in thecontext of the present invention that the membrane or layer permits thepassage of both liquid and vapor phases. Such hydrophilic layers willalso be referred to herein as liquid permeable membranes. Where liquidpermeable membranes are provided, passage of the fluid phase into thedischarge zone is permitted.

The concentration patch is preferably provided with an analytedetermination zone having detection chemistry such as an immobilizedspecific binding partner for an analyte to be determined in the bodyfluid. An analyte reference zone may additionally be provided, whichprovides a means for determining whether a sufficient amount of bodyfluid has passed through the analyte determination zone to sufficientlydetermine the existence of the analyte. The analyte reference zonepreferably produces a visible indicium upon exposure to a predeterminedthreshold, such as a predetermined volume of fluid, or a thresholdamount of a reference analyte such as IgG, albumin or the like.

In accordance with a further aspect of the present invention, there isprovided a method of detecting false negative results in an assay of abody fluid from a subject, which are the result of noncompliance withthe testing procedure by the subject. The method comprises the steps ofsecuring a test patch to the subject in communication with a source ofbody fluid, the test patch comprising a first detection chemistry fordetecting the presence of an analyte in the body fluid, and a seconddetection chemistry for detecting the presence of a referencesubstituent in the body fluid.

The test patch is removed from the subject after a sufficient testperiod of time to enable the first detection chemistry to detect theanalyte, if present in the body fluid. The amount of referencesubstituent detected by the second detection chemistry is thendetermined, and the amount of reference substituent determined iscompared to a predetermined value to determine whether the test patchwas actually worn for substantially all of the test period.

Further features and advantages of the present invention will becomeapparent from the Detailed Description of Preferred Embodiments whichfollows, taken together with the claims and appended figures hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a dermal concentration patch accordingto one embodiment of the present invention.

FIG. 1a is a cross-sectional view along the line 1a--1a of the dermalconcentration patch of FIG. 1.

FIG. 2 is a perspective view of a dermal concentration patch accordingto a second embodiment of the present invention.

FIG. 2a is a cross-sectional view along the line 2a--2a of the dermalconcentration patch of FIG. 2.

FIG. 3 is a perspective view of a third embodiment of the dermalconcentration patch of the present invention.

FIG. 3a is a cross-sectional view along the line 3a--3a of the patch ofFIG. 3.

FIG. 4 is a perspective view of one embodiment of a reagent packet foruse in effecting a color change responsive to the presence of analyte inthe concentration patch of the present invention.

FIG. 5 is an exploded elevational schematic view of a fourth embodimentof the present invention.

FIG. 6 is a cross-sectional view of a dermal patch according to afurther embodiment of the present invention.

FIG. 7 is a plan view of a dermal patch according to another embodimentof the present invention.

FIG. 8 is an exploded elevational view of a dermal concentration patchaccording to yet another embodiment of the present invention.

FIG. 9 is a plan view of a dermal concentration patch according to afurther embodiment of the present invention.

FIG. 10 is an exploded elevational view according to still anotherembodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to FIG. 1, there is disclosed a dermal concentration patch 10according to one embodiment of the present invention, illustrated assecured to the surface of the skin 12. As will be appreciated by one ofskill in the art, the concentration patch of the present invention maybe used for veterinary purposes as well as on humans. In addition, theconcentration patch can be used in more diverse applications such as inagriculture or any other environment where a chemical species is to bedetected in a fluid and a heat source such as body heat, sunlight, etc.is adaptable to effectuate the distillation or other concentrationfunction of the patch. The preferred use, however, is for determinationof preselected chemical species in sweat, and the ensuing discussion isprincipally directed to that end use.

Moisture expressed from the skin 12 within the perimeter of the testpatch 10 first accumulates in a concentration zone 14 beneath the firstside of a gas permeable filter 16. The concentration zone 14 preferablycontains a fluid-permeable medium 20 which may be cotton guaze or othercommonly available permeable material. For example, a layer of any of avariety of known fiber webs such as knitted fabrics, or nonwoven rayonor cellulose fibers may be used. Filtration Sciences #39 is aparticularly preferred fluid-permeable medium for use as a concentrationzone in the present invention.

Moisture accumulates in the interfiber spaces of the medium 20 and,under the influence of body heat which is readily conducted from thesurface of the skin through the fluid phase, the water component of theperspiration will tend to volatilize.

As previously discussed, the concentration patch 10 is provided with agas permeable filter 16. By "gas permeable," I intend to designate anymaterial which will permit the passage of the vapor phase of fluidsexpressed from the skin, but substantially retain the fluid phase withinconcentration zone 14. Any of a variety of suitable commerciallyavailable microfiltration membrane filters may be used for this purpose,such as the Gore-Tex 0.45 micron Teflon filter manufactured by W. L.Gore & Associates, Inc. (Elkton, Md).

I use the term "liquid permeable" in this patent to mean a materialwhich will permit the passage of sweat in the liquid phase. A liquidpermeable filter will allow the passage of water in both the liquid andvapor phases. Thus, when the term "water" is used herein, I mean torefer to both the liquid and vapor phases of water, unless reference isspecifically made to a particular phase.

Adjacent the second side of the gas permeable filter 16 is a dischargezone 18. As previously discussed, gas permeable filter 16 retains thefluid phase but permits escape of the vapor phase of the fluid componentin perspiration. Thus, the vapor component which primarily consists ofvaporized water continuously escapes through the gas permeable filter 16exiting the second side thereof into discharge zone 18, which is incommunication with the atmosphere. In an alternative embodiment, notseparately illustrated, the gas permeable filter 16 is replaced by aliquid permeable membrane which permits passage of the fluid phase. Inthis embodiment, fluid, or a combination of vapor and fluid, will bepermitted to escape from the concentration patch. Any of a variety ofliquid permeable filters are commercially available which can be used toform a liquid permeable filter used in this embodiment of the presentinvention. A preferred liquid permeable filter is constructed from JamesRiver Paper Drape.

Disposed adjacent the second side of filter 16 in the discharge zone 18is a flexible permeable outer layer 22. This layer serves to protect thefilter 16 against physical damages such as abrasion, and can also serveas a barrier for preventing chemical contamination of the filtermaterial from the outside. Layer 22 may comprise any of a variety ofcommercially available vapor permeable tapes and films which are knownto one of skill in the art. Layer 22 may be distinct from or integralwith tape 26 discussed below. Alternatively, depending upon the intendedapplication of the patch, layer 22 may be deleted altogether, where itdoes not appear that abrasion or external contamination willdeleteriously affect the concentration patch 10.

The concentration patch 10 illustrated in FIG. 1 is secured to thesurface of the skin by means of a peripheral band of tape 26.Preferably, tape 26 will extend around all sides of patch 10. Forexample, an annular ring of tape can be die punched for use with acircular patch, or the center of a rectangular piece of tape can beremoved to expose layer 22 or filter 16 of a rectangular patch. SeeFIGS. 1 and 3, respectively. Alternatively, layer 22 and tape 26 couldbe deleted altogether and layers 16 and 20 could be secured to thesurface of the skin by a bandage. One such method would be to capturelayers 16 and 20 under a bandage or wrapping surrounding the arm or theleg. In this case, the vapor and/or fluid is permitted to escape through16 and 20 and into the bandage where it may either collect there ordissipate into the environment.

A large variety of hypoallergenic or other suitable tapes are well knownin the art, which may be adapted for use with the concentration patch 10of the present invention. Different tapes or adhesives may be desirabledepending upon the intended use of the test kit, based upon theirability to adhere to the skin during different conditions such asdaytime wearing under clothing, during sleep, during profuse sweatingfor prolonged periods or during showers. It has been determined that themost desirable tapes include multiple perforations which prevent sweatfrom building up underneath the tape and eventually compromising theintegrity of the adhesive. Preferably, a tape, such as Dermiclearmarketed by Johnson & Johnson, will be used.

Any of a wide variety of means for securing the concentration patch 10to the skin 12 may be utilized. For example, the tape 26 can beeliminated and gauze layer 20 provided with a lower adhesive layer toperform the same function. One such adhesive membrane is the MN-100adhesive membrane manufactured by Memtec of Minnetonka, Minn. Thismembrane is liquid permeable so that it passes fluid as would the gauzelayer 20, yet has one adhesive side so that it will stick to the skin.Alternatively, outer protective layer 22 can comprise an annular flange23, extending radially outwardly beyond the outer edges of filter 16 andgauze 20. See FIG. 2a. The lower surface of the flange 23 is thenprovided with a suitable adhesive.

The surface temperature of human skin varies regionally, however, it isgenerally within the range of from about 86° to about 90° F. at rest,and can rise to much higher temperatures under conditions of strenuousexertion. At those temperatures, a number of chemical species ofinterest for the purpose of the present invention, such as creatinekinase, a high or low density lipoprotein have a sufficiently low vaporpressure that volatilization is not a significant factor in theefficiency of the concentration function. At the same time, thesubstantial aqueous component will have a sufficiently high vaporpressure that it will tend to volatilize thereby concentrating the lessvolatile fractions. However, in some applications, the chemical speciesof interest will have a high enough vapor pressure, even at the restingtemperature, such that escape of the vapor phase through the gaspermeable filter 16 of the analyte of interest will disadvantageouslyimpair the efficacy of the test patch. For these analytes, a modifiedconcentration patch must be used.

Referring to FIGS. 2 and 2a, there is disclosed a modified concentrationpatch 11 according to the present invention for use with an analytehaving a propensity to escape through the gas permeable filter 16 as avapor under ordinary use conditions. The test patch comprises aconcentration zone 14 defined on its inner boundary by the skin 12 towhich the concentration patch 11 is secured. The outer boundary of theconcentration zone 14 is defined by gas permeable filter 16, whichseparates the concentration zone 14 from the discharge zone 18. Disposedin the concentration zone 14, and adjacent the gas permeable filter 16,is a binder layer 31 for binding and preventing the escape of moleculesof the volatile analyte. The binder layer 30 is separated from the gauzelayer 20 by a porous layer 28, which may compromise any of a variety offilms for retaining the binder layer 31 yet permitting passage of fluid.

In the embodiment illustrated in FIG. 2a, perspiration will pool in theinterfiber spaces of the gauze 20, and will percolate through porouslayer 28 into the binder layer 31. In that layer, a chemically active orbiochemically active binder material will act to selectively bind thevolatile analyte, thereby preventing it from escaping as a vapor throughgas permeable filter 16. As discussed in connection with the embodimentillustrated in FIG. 1, it is also possible to replace the gas permeablefilter 16 with a liquid permeable layer, where the presence of fluid onthe outside of the test patch would not be undesirable.

The binder layer 31 may comprise any of a variety of binders dependingupon the nature of the volatile analyte to be determined. For example,the binder may chemically bind with the analyte or adsorb the analyte tobe determined. In addition, the binder layer may comprise a specificbinding partner of the analyte to be determined, such as a polyclonal ormonoclonal antibody or an antigen matched to a specific antibody desiredto be measured in the perspiration.

The concentration patch 11 is additionally provided with tape 26 oranother means for securing the patch to the skin of a subject, as hasbeen detailed in connection with the embodiment illustrated in FIG. 1.Concentration patch 11 is illustrated, however, as having a unitaryouter layer 22 extending beyond the perimeter of the underlying layersto form an annular flange 23, which is provided with an adhesive on itslower surface. As discussed in connection with the embodiment of FIG. 1,outer protective layer 22 permits the escape of water vapor yet protectsthe filter material from chemical contamination from the outside.

Referring to FIGS. 3 and 3a, there is disclosed a further embodiment ofthe test patch of the present invention wherein an inner porous layer 28and an outer porous layer 30 define a space for containing a microbeadlayer 32. The inner layer 28 and outer layer 30 preferably comprise thesame material, which may be any suitable material for providing anunrestricted flow of fluid through the patch while trapping themicrobeads in between. One suitable material for porous layers 28, 30 isthe liquid permeable and microporous film known by the name Ultipor(nylon 6) and manufactured by Pall Corporation in Glen Cove, N.Y.Additional manufacturers of suitable nylon filtration membranes includeMicron Separations, Inc. of Westborough, Mass., and Cuno of Meridan,Conn. Porous layers 28, 30 may also be comprised of materials other thannylon, such as polycarbonate, modified polyvinylchloride andpolysulphone.

The gauze, the inner and outer porous layers and the adhesive tape inall embodiments could be cut to size with conventional dies. The gauze20 and the inner porous layer 28 could be fixed to the adhesive ring 26with conventional adhesives, such as used on the adhesive surfaceitself. Alternatively, they could be heat or ultrasonically bondedtogether. The proper amount of microbeads could then be placed on top ofthe inner porous layer and then the outer porous surface attached bysimilar means. Typically, in a one-inch diameter patch, from about 0.05grams to about 1 gram of microbeads will be used, and preferably fromabout 0.1 to about 0.4 grams will be used. The inner and outer poroussurfaces may have to be staked or spot-welded together in some pattern,as will be appreciated by one of skill in the art to prevent themicrobeads from collecting in one area.

The free adhesive surface is optimally covered by pull-away paper (notillustrated) with adequate space to be gripped with one's fingers. Thepatch is packaged in a paper or plastic pouch similar to that used inconventional band-aid packaging. The assembled unit could be terminallysterilized or pasteurized prior to sale. Alternatively, the packagecould comprise a vapor barrier such as a metallic foil or mylar and eveninclude oxygen or moisture absorbent means such as a small packet of anyof a variety of known desiccants, such as silica gel, calcium chloride,calcium carbonate, phosphorous pentoxide or others as will beappreciated by one of skill in the art.

The total thickness of microbead layer 32 can be varied considerably.However, if the color change is to be developed by immersion of thepatch in appropriate reagent baths, layer 32 is preferably no more thanabout 3 mm thick since color changes occurring at immobilized sites onthicker layers would not likely be observable. More preferably, themicrobead layer is between about 1 mm and about 2 mm thick.Alternatively, microbead layer 32 could be torn open, releasing loosemicrobeads which could be used to conduct chemical analysis for presenceof the unknown by conventional means, such as in a cuvette.

Optimally, the diameter of the beads in microbead layer 32 will be atleast about one order of magnitude larger than the diameter of the poresin inner porous layer 28 and outer porous layer 30. For example, thebeads contained in microbead layer 32 may have diameters within therange of from about 5 to 50 microns, and preferably within the range offrom about 5 to about 10 microns. If 10-micron diameter beads areutilized in the microbead layer 32, for example, inner porous layer 28and outer porous layer 30 will optimally comprise a median pore size ofapproximately 1 micron.

Microbead layer 32 may comprise any of a variety of known materialsincluding polystyrene, latex and glass. Beads sized from approximately0.05 micron to 100 micron which are suitable for the present applicationare available from Polysciences of Warrington, Pa.

Microbead layer 32 serves as the support for an immobilized specificbinding partner for the analyte to be determined. Thus, a molecule witha high chemical affinity for a specific component in the fluid to beanalyzed will be immobilized to the microbeads in microbead layer 32.

Referring to FIG. 5, there is disclosed a further embodiment of thepresent invention, particularly suited for use under conditions in whichprofuse sweating is not present, such as in passive insensibleperspiration, wherein the test patch is provided with an impermeableouter layer 42. In order to minimize any back diffusion of fluid intothe skin, an absorptive layer 44 is provided to form a reservoir fordrawing moisture away from the surface of the skin and through support46 to which is bound a specific binding partner for at least one analyteto be determined. Layer 44 may include chemical means for binding orcollecting moisture such as a desiccant as has been previouslydiscussed, which is suitable for use in proximity to the skin. The patchmay be further provided with an underlying porous layer 48 to separatesuppport 46 from the surface of the skin, and the patch is provided withany of the means for attachment to the skin as have been previouslydiscussed.

In one preferred embodiment of the present invention, the analyte to bedetermined in perspiration is the enzyme creatine kinase MB (CK-MB)which is expressed from the cardiac muscle during myocardial infarctionand other cardiac distress. A monoclonal antibody raised against CK-MBcan be immobilized to the microbeads in accordance with any of a varietyof conventional methods, such as the cyanogen bromide techniquedescribed in Pharmacia product literature (Pharmacia, Inc., Piscataway,N.J.).

The monoclonal antibodies useful in the present invention may beproduced and isolated by processes which are well known in the art, suchas those discussed by Milstein and Kohler, reported in Nature, Vol. 256at 495-497 (1975). In particular, Jackson describes a method ofproducing anti-CK-MM (an indicator of the status of skeletal muscles)and anti-CK-MB antibodies in Clin. Chem., 30/7, 1157-1162 (1984).

In accordance with one known process, mice such as Balb/c female mice orother mouse strains or even other suitable animals such as rats orrabbits are immunized with an amount of the CK-MB enzyme to initiate animmune response. The enzyme dosage and immunization schedule forproducing useful quantities of suitable splenocytes can be readilydetermined depending on the animal strain used.

The size and spacing of doses of CK-MB or other antigen are of primeimportance in the antibody response. Fortunately, a wide range ofantigen doses commonly affords immunity againts harmful agents. Thus, asmall dose of antigen is usually sufficient to initiate an antibodyresponse, i.e., microgram quantities of proteins are frequentlyadequate. However, a minimum dosage for initiating an immune responsedoes typically exist, although doses of antigen below the minimum dosenecessary to initiate an antibody response will usually maintainantibody production which is already in process. For example, an initialimmunization with approximately 50 μg of the enzyme may be followed by ahyperimmunization series of five injections.

When certain compounds which are themselves not necessarily antigenicare mixed with an antigen, enhanced antibody production against theantigen occurs, as evidenced by the appearance of large amounts ofantibody in the serum, a prolonged period of antibody production, and aresponse to lower doses of antigen. Such substances are called"adjuvants" and include Freun's incomplete and complete adjuvants andalum gels. Thus, a given dose of antigen is usually more effective wheninjected subcutaneously with an adjuvant or when injected as repeatedsmall aliquots than when administered intravenously.

Typically, the adjuvants of Freund are preferred. The original"complete" Freund's adjuvant mixture consists of mineral oil, waxes andkilled tubercle bacilli. Antigen is added to the adjuvant mixture in anaqueous phase to form a water-in-oil emulsion in which each waterdroplet is surrounded by a continuous oil phase containing tuberclebacilli. The mixture is commonly injected subcutaneously intoexperimental animals. Injection stimulates a marked granulomatousreaction with lesions consisting largely of collections of histiocytes,epithelioid cells and lymphocytes. The local lymph node shows a smallincrease in plasma cells.

Following the immunization with a primary dose of a soluble proteinantigen, specific antibodies normally first appear in the serum after afew days increase until about the second week, and thereafter, slowlydecline over a period of weeks to months.

The first serum antibodies to appear after antigenization are IgMantibodies. These are usually followed by the appearance of IgGantibodies. Later, as antibody serum levels increase, IgM antibodiesdisappear, probably as a result of specific feedback suppression of IgGantibodies.

After the "primary response" to a protein has passed, a second dose ofthe same antigen given months or even years later usually elicits anintense and accelerated "specific secondary response" in which serumantibody usually begins to rise within two or three days of exposure.The serum levels of antibody in a secondary response may reach as highas 10 mg per ml.

The animal is subsequently sacrificed and cells taken from its spleenare suspended in an appropriate medium and fused with myeloma cells,such as those obtainable from the murine cell line Sp2/0-Ag14. Theresult is hybrid cells, referred to as "hybridomas," which are capableof reproduction in vitro and which produce a mixture of antibodyspecific to each of the various recognizable sites on the CK-MB enzyme.

The myeloma cell line selected should be compatible with the spleencells, and optimally of the same species. Although the murine cell lineSp2/0-Ag14 has been found to be effective for use with mouse spleencells, other myeloma cell lines could alternatively be used. See, forexample, Nature, Vol. 276 at pp. 269-270 (1978).

The myeloma cell line used should preferably be of the so-called "drugresistant" type, so that any unfused myeloma cells will not survive in aselective medium, while hybrid cells will survive. A variety of drugresistant myelomas are known.

The mixture of unfused spleen cells, unfused myeloma cells and fusedcells are diluted and cultured in a selective medium which will notsupport the growth of the unfused myeloma cells for a time sufficient toallow death of all unfused cells. A drug resistant unfused myeloma cellline will not survive more than a few days in a selective medium such asHAT (hypoxanthine, aminopterin and thymidine). Hence, the unfusedmyeloma cells perish. Since the unfused spleen cells are nonmalignant,they have only a finite number of generations until they fail toreproduce. The fused cells, on the other hand, continue to reproducebecause they possess the malignant quality contributed by the myelomaparent and the enzyme necessary to survive in the selected mediumcontributed by the spleen cell parent.

The supernatant from each of a plurality of hybridoma containing wellsis evaluated for the presence of antibody to a specific site unique tothe CK-MB enzyme structure. Hybridomas are then selected producing thedesired antibody to that specific site. This selection may be, forexample, by limiting dilution, in which the volume of diluent isstatistically calculated to isolate a certain number of cells (e.g., 1to 4) in each separate well of a microliter plate. In this way,individual hybridomas may be isolated for further cloning.

Once the desired hybridoma has been selected, it can be injected intohost animals of the same species as those used to prepare the hybridoma,preferably syngeneic or semi-syngeneic animals. Injection of thehybridoma will result in the formation of antibody producing tumors inthe host after a suitable incubation time, resulting in a very highconcentration of the desired antibody in the blood stream and in theperitoneal exudate of the host. Although the hosts have normalantibodies in their blood and exudate, the concentration of these normalantibodies is only about 5% of the concentration of the desiredmonoclonal antibody. The monoclonal antibody may then be isolated inaccordance with techniques known in the art.

Alternatively to raising anti-CK-MM monoclonals as described, thecomponents of a commercially available diagnostic kit could be utilized,which incorporates the CK-MM enzyme chemically bound to a bead support.A suitable kit marketed as the Isomune-Ck Diagnostic Kit by Roche ofNutley, N.J., is one commercially available candidate. This kit includesa goat antisera to human CK-MM and donkey anti-goat antibody covalentlybound to styrene beads. A mixture would produce an immobilized conjugatehaving a specific affinity for human CK-MM. A more direct and lessexpensive procedure, however, would be to immobilize the anti-CK-MMmonoclonal antibody directly to the microbead support in accordance withmethods now well known in the art.

The antibody which is to be used for the purpose of complexing withCK-MB may be immobilized on any of a variety of supports known in theart. For example, anti-CK-MB antibody may be bound to polysaccharidepolymers using the process described in U.S. Pat. No. 3,645,852.Alternatively, the antibody may be bound to supports comprising filterpaper, or plastic beads made from polyethylene, polystyrene,polypropylene or other suitable material as desired. Optimally, thesupport will take the form of a multiplicity of microbeads which canconveniently be formed into microbead layer 32, illustrated in FIG. 3a.

As an alternative to a microbead support layer, the specific bindingpartner could be immobilized directly to the inner porous layer 20 or 28on FIG. 3a or to the underside of filter 16 of FIG. 1a. In this manner,the need for microbead layer 32 could be eliminated entirely. Liquidpermeable membranes which are specifically designed for binding antibodyproteins are commercially available, such as Zetapor from Cuno, andProtrans, available from ICN in Costa Mesa, Calif.

Referring to FIG. 4, there is disclosed a reagent packet for use withthe concentration kit of the present invention. The reagent packet 34comprises a container 36 having a removable secured top 38. A flap 40 onthe top 38 facilitates gripping the top 38 and peeling away fromcontainer 36 to reveal the reagent contained therein.

Typically, after the test patch has been worn for a suitable period oftime, it will be removed by the wearer (in non-drug screen tests) anddeveloped to produce a visible indicium of the test result. The testpatch can be marketed together with a developer packet such as packet 34which contains known developer reagents for the immunoassay. Forexample, a protein electrophoresis stain such as Coomassie brilliantblue or amido black 10b, can be bound to purified analyte contained inthe reagent packet 34. When a test patch is immersed in the packet 34,any antibodies on the test patch that are unbound by analyte in theperspiration will become occupied by stained purified analyte in thepacket 34. There will thus be an inverse relationship between the amountof stain absorbed by the patch and the amount of enzyme passed throughthe patch. In this embodiment, the user would place the patch in thefluid of the packet 34, wait for some period of time such as 30 secondsor more, rinse the patch under tap water and relate the resultant colorof the patch to the presence of the enzyme. A color comparison chart andcontrol zone on the patch having no bound antibody may be provided toaid in this interpretation.

Alternatively, the user could support the test patch on an open vessel,such as a small jar or vial, or empty container similar in design toreagent packet 34 securing the adhesive border of the patch to the rimof the vessel, and then pour contents of packet 34 on top of the testpatch. Gravity would assist the transport of the contents of packet 34through the test patch to maximize the efficiency of the stain/bindingreaction, and to facilitate visualization of the color change.

The system could readily be designed so that the user performs theinterpretation of the concentration of the analyte not in the patch atall but by observing the packet contents once the contents havetraversed the patch. This method would be similar to conventional ELISAassay methods where the packet contents contain enzyme conjugates whichwill react to specific enzyme substrates. The enzyme substrates would beadded to the packet contents after those contents transversed the testpatch.

If the perspiration contained molecules of interest, they would bind tothe specific immobilized binding partner on the patch. If this occurred,enzyme conjugates in the packet would pass freely across the test patchand enzymatically modify the enzyme substrate producing a controlledcolor change in the solution in the packet. If the perspirationcontained the desired molecules of interest, enzyme conjugates wouldthen be bound in transit across the patch and would be unavailable tocause color change in the substrate solution. Other immunoassay schemescan be readily adapted for use in the present invention by one of skillin the art.

Although the concentration patch of the present invention could be usedfor any of a variety of body fluids, perspiration is the desired fluiddue to its dependable supply and its similarity to blood, albeit withlower analyte concentrations. Saliva also appears to contain many of thechemical components of blood, however, often at even lesserconcentrations than found in sweat.

In performing the method of the present invention, the concentrationpatch may advantageously be located on different regions of the bodydepending upon a variety of factors. It is well known that the quantityof perspiration generated is a function of both the location on thebody, as well as the physical activity during and immediately precedingcollection. This is due to both different densities of sweat glands ondifferent regions of the body, as well as to certain regulatoryfunctions of those glands.

Sweat glands are classified to be either of two types. Eccrine typefunction primarily to regulate body temperature through theirrelationship to evaporative heat loss. It is the eccrine type sweatgland that provides the sweat associated with exercise and is thereforethe source of perspiration of interest for many applications of theconcentration patch of the present invention. Apocrine type sweat glandsare larger secreting elements which are localized only in relativelyisolated areas of the body such as the axilla, pubic and mammary areas.

Although the etiology of perspiration is relatively complex, it is knownto be caused by both mental states such as mental exercise and emotionalstress; thermal stress, as the sedentary body's response to temperaturecontrol; and exercise stress as the physically active body's response totemperature control.

In addition to the foregoing distinctions, perspiration can be eitherinsensible or sensible. Insensible sweat appears to be caused by waterdiffusion through dermal and epidermal layers. Its purpose appears to benot related to thermal regulation at all, but to aid in such things asthe improvement of mechanical interaction between the skin and surfacesto facilitate grip. Further complexities arise with regard to thespatial distribution of sweat glands and the flow rates of the varioustypes of perspiration. Specialized areas of the palms and soles of thefeet sweat continuously, although at a very low rate. The rate ofinsensible perspiration is dependent upon the position of the particulararea in question relative to the heart. For example, elevating a limbover the heart decreases the insensible perspiration rate.

At temperatures of less than about 31° C. in a resting human adult,insensible perspiration proceeds at a rate of between about 6-10 gramsper square meter per hour from the skin of the arm, leg and trunk, up toabout 100 grams per square meter per hour for palmer, planter and facialskin. The latter three areas jointly account for approximately 42% ofthe total water loss from the body under non-sweating conditions, whichgenerally means an air temperature of between about 24°-26° C. Suchinsensible perspiration first begins on the dorsal surfaces of the footand spreads to higher places on the body as the temperature increases.One reported study determined that the average water loss due toinsensible perspiration for a body surface area of 1.75 square metersranged from 381 ml, 526 ml and 695 ml per day at ambient temperatures of22° C., 27° C. and 30° C., respectively.

In contrast to insensible perspiration which does not appear to beassociated with a particular surface element of the skin, sensibleperspiration has been associated with the eccrine gland. The number ofactively secreting eccrine glands varies among individuals and dependsupon the part of the body observed and the type of sweat responsecreated. Maximum gland density varies from between about 200 per squarecentimeter on the forearm to over 400 per square centimeter on thethenar eminence.

The appearance of sensible sweat begins at either when the skintemperature exceeds about 94° F. or the rectal temperature exceeds about0.2° F. over normal core temperature. Maximum rates of sweat volume losscan be as high as 2 liters per hour in average subjects and can be ashigh as 4 liters per hour for brief periods. Sensible perspirationbegins in the distal parts of the lower extremities and progressesupward as the environmental temperature is elevated. Thus, the dorsum ofthe foot begins to sweat long before the chest. The pattern of sensiblesweat response also shifts from one region of the body to another as thethermal stress increases. Under mild thermal stress, sweating is presentmainly in the lower extremities. As the thermal stress furtherincreases, sweating spreads to the trunk. Due to its large surface area,the trunk becomes the dominant water loss surface. Eventually, extremelyhigh rates are found in the trunk while rates in the lower extremitiesmay actually decline. The forehead can produce extremely high sweatrates but is among the last areas to sweat in response to thermalstress.

As has been described previously, a large variety of chemical specieswhich are detectable in blood are also present in sweat, althoughtypically in a much lesser concentration. Early investigation into thecomposition centered on electrolytes, including sodium, chloride,calcium and potassium. Extreme individual variation was found amongindividuals, and the electrolyte composition also differed dependingupon whether the sweat was stimulated by thermal, mental or otheretiology.

Further research has identified numerous additional components in sweat,including both electrolytes and more complex biological molecules. Someillustrative chemical species which have been identified in sweat areidentified in Table I below.

                  TABLE I                                                         ______________________________________                                        Chemical Components of Sweat                                                  ______________________________________                                        diphtheria antitoxin                                                                              sulfates                                                  ascorbic acid       iodine                                                    thiamine            iron                                                      riboflavin          fluorine                                                  nicotinic acid      bromine                                                   amino acids         bismuth                                                   ethanol             lactic acid                                               antipyrine          pyruvate glucose                                          creatinine          nitrogen                                                  C-14 methylurea     ammonia                                                   C-14 acetamide      uric acid                                                 C-14 urea           nicotine                                                  thiourea            morphine                                                  paraaminohippuric acid                                                                            sulfanilamide                                             mannitol sucrose    atabrin                                                   lactate             methadone                                                 sodium chloride     phencyclidine                                             potassium           aminopyrine                                               calcium             sulfaguanidine                                            magnesium           sulfadiacine                                              phosphorous         amphetamines                                              manganese           benzoylecgonine                                           theophylline        phenobarbital                                             parathion           androgen steroids                                         tetrahydrocannabinol                                                                              phencyclidine                                             insulin             phenytoin                                                 cimetidine          carbamazepine                                             dimethylacetamide                                                             ______________________________________                                    

Any of the entries in Table I for which affinity chemistry can bedeveloped, can be an appropriate subject of a test patch according tothe present invention. Since most of the components listed in Table Iare non-volatile, they will be trapped in the concentration zone 14 ofthe concentration patch 10 illustrated in FIG. 1a, or on the binderlayer 30 of FIG. 6. However, some components, most notably ethanol,would volatilize under the influence of body heat thereby enablingescape in the vapor phase through the test patch. Where the analyte tobe determined is ethanol or another volatile component, theconcentration patch of the present invention may be modified asdescribed in connection with the embodiment illustrated in FIG. 2.

Referring to FIG. 6, there is disclosed a modified concentration patch13 according to the present invention, in which all intervening layersbetween the skin 12 and the binder layer 30 have been deleted. Bydisposing the binder layer (i.e., the layer having a specific bindingpartner for an analyte to be determined) directly adjacent the skin,lateral diffusion of sweat throughout the binder layer 30 is minimized.The proximity of the binder layer 30 to the skin 12 allows the output ofeach duct of the sweat glands to contact or be in fluid communicationwith a relatively small area of the binder layer 30. For a variety ofreasons which will be apparent to one of skill in the art, it may alsobe desired to mount a microporous membrane, preferably a liquidpermeable membrane 50 atop the binder layer 30.

The evaporative capacity of the binder layer 30 and the liquid permeablemembrane 50 is preferably sufficient relative to the output capacity ofthe individual sweat ducts, to minimize lateral diffusion of sweat awayfrom the immediate area of the duct. This embodiment has specialapplication for monitoring the chemical composition of insensibleperspiration and/or non-exercise perspiration, in instances where outputfrom the sweat glands is limited. Due to the magnification effectdetailed infra, the present embodiment is also particularly suited formonitoring low concentration analytes.

By limiting the suppressive characteristics of moisture or water on theskin, through the use of materials having a maximal evaporativecapacity, the instant embodiment allows increase of the through-put rateof sweat in the patch by maximizing sweat gland output. Nadel andStolwijk (J. Applied Physiology, 1973, 35(5); 689-694) disclose thatsweat gland activity is suppressed by water lying on the skin, finding adifference in whole body sweat rate of 40% between wet and dry skin.Mitchell and Hamilton (Biological Chemistry, 1948, 178:345-361), foundthat loss of water and solutes in insensible perspiration presumablystops whenever the surface of the skin is covered with a film of water.Brebner and Kerslake (J. Physiology, 1964, 175:295-302) postulate thatthe reason for this phenomenon is that water in contact with the skincauses the epidermal cells of the skin to swell and thus block the sweatducts.

The ability of the present invention to produce a positive responsebased upon the presence of relatively low concentrations of analyte isparticularly advantageous in view of the fact that, during activeexercise, a 1/4" diameter area of skin provides approximately 35microliters of sweat per hour, whereas a similar diameter area of skinproduces sweat at a non-exercise rate of only about 3.2 microliters perhour. The present embodiment is further advantageous as not requiringthe user to exercise, but only to wear the patch for an equal ortypically longer period during rest or at normal activity levels.

Thus, homogeneous diffusion of sweat throughout the binder layer ispreferably minimized when using the instant invention in conjunctionwith insensible and/or non-exercise perspiration and/or a determinationof minute amounts of analyte contained within perspiration. Theminimized lateral diffusion of perspiration throughout the binder layer30, according to the present invention, provides a more concentratedcollection of sweat at each sweat duct, thereby providing a greateramount of selected analyte to be determined at that area.

Sato and Fusako (American J. Physiology, 1983, 245(2): 203-208) estimatethat the diameter of the duct of the sweat gland is approximately 40microns. According to Scheupoein and Blank (Physiological Review, 1971,51(4): 702-747), the average density of sweat glands on the skin surfaceis approximately 250 per square centimeter. Thus, the total surface areaof sweat gland ducts of the skin represent 1/318 of the total surfacearea of the patch of the instant invention. The visible result on a testpatch of the present invention when, for example, using known ELISAtechnology to determine a low concentration analyte, is the appearanceof a number of tiny color changes on the binder layer 30 associated withthe output of specific ducts. If significant lateral diffusion of sweatis permitted prior to contact with the immobilized binding partner, thecolor change is frequently too diffuse to detect with the naked eye.

Although a patch incorporating the present embodiment of the inventionmay be worn at any practical location on the body, preferable locationsfor the patch include the skin on the sole of the foot, and areas on thechest and back. The patch is able to be worn in confidence in theseareas, and these areas are not covered with excessive hair, so that thepatch may be secured with conventional adhesive tapes.

Hertzman, et al. (J. Applied Physiology, 1952(5): 153-161) determinedthat the rate of sweat output from the sole is independent ofenvironmental temperature, and, from this, it is suspected that it isalso independent of activity level. Therefore, as the sole does notgenerate exercise sweat, the input to a patch on this location would beindependent of the activity level of the wearer.

Referring to FIG. 7, there is shown a modified binder layer 52 for aconcentration patch according to the present invention, wherein two ormore distinct zones are provided on the binder layer 52. The use of areference zone or of several distinct test zones is contemplated forboth the single layer patch discussed in connection with FIG. 6, as wellas the embodiments discussed in connection with FIGS. 1-3a and 5. Themulti-zone binder layer 52 may also be used for certain embodiments tobe discussed hereinafter in connection with FIGS. 6-10 when specificbinding chemistry is used.

One or more of the zones, such as determination zone 60 (FIG. 7), isused to test for an analyte of interest within the sweat, as detailedpreviously. One or more of the remaining zones, such as reference zone61, is used as a reference indicator.

Reference zone 61 performs a variety of functions, depending upon thedesired application of the test patch. For example, reference zone 61can be provided with color change chemistry as discussed previously toprovide the wearer with an indication that the patch has been worn forlong enough that a sufficient sample volume has traversed the patch toprovide a meaningful test for the analyte of choice. For this purpose,reference zone 61 is provided with affinity chemistry for a preselectedreference substituent such as IgG, albumin or any other sweat componentwhich is reliably present. Preferably, the selected referencesubstituent is one which provides a reasonably accurate measurement ofthe volume of sweat put through the system.

This use of the reference zone 61 may be facilitated by firstdetermining the rough concentration ratio of a reference substituentsuch as albumin to the analyte to be determined and providing the patchwith color change chemistry which provides a visual indication of thepresence of the reference substituent only well after the elution of theanalyte to be determined has exceeded the lower limits of detection.

Reference substituents such as albumin will typically be present insignificantly greater quantities than the analyte. Thus, in order toaccomplish the objective of indicating passage of a sufficient samplevolume, the "sensitivity" of the patch for the reference substituent ispreferably lower than for the analyte. This can be achieved by using aproportionately lower amount of specific binding partner for thereference substituent than for the analyte, other dilutions in theassay, or simply selecting a less abundant reference substituent.Selection of a suitable reference substituent and concentrationdeterminations can be readily made through simple experimentation by oneof skill in the art.

Alternatively, and particularly useful in assays for drugs of abuse andtheir metabolites, a reference zone 61 can provide an indication thatthe skin patch was actually worn by the desired patient, parolee orother subject. One inherent limitation in a test in which a subjectdesires a negative result is the possibility that the subject willsimply remove the patch after administration and replace it just priorto reexamination. This possibility gives rise to the ability of thewearer to ensure false negative results.

However, by provision of a reference zone 61 to detect a known componentin sweat, the test results will reveal test patches that have not beenworn for the test period. Reference zone 61 thus provides a method ofpreventing false negative evaluations due to tampering or removal of thetest patch.

A reference zone 61 to detect a known component in sweat may also beprovided as a positive control zone to ensure the discovery of falsenegative test results due to degradation of reagents or other componentsof the patch.

In non drug-of-abuse screens, the indication produced within thereference zone 61 will preferably be a visible color change by achemical or antibody/antigen colorimetric interaction occurring orbecoming apparent to the wearer when a predetermined amount of thereference analyte has passed through the interaction area.

Optionally, a reference zone 61 may be provided as a negative controlzone to enable the discovery of false positive results. A preferrednegative control zone will have an immobilized specific binding partnerfor an analyte known to be absent in human sweat. The analyte's specificbinding partner must be known to not cross react with components presentin human sweat. An example of an appropriate analyte is bacteriophage T4coat protein.

In yet a further embodiment of the present invention (not illustrated)two or more analyte determination zones 60 are provided in a single testpatch. The use of multiple test zones is particularly useful inapplications such as a drug of abuse screen where testing for any one ormore of a wide variety of analytes may be desired. For example, a singletest patch may be used to screen for any of a plurality of drugs ofabuse, such as THC, Phencyclidine morphine and Methadone. A positiveresult for any of the drugs on the screen may provide sufficient proofof an offense such as a violation of parole, or can be used to signalthe need for more quantitative follow up investigations. Used as aninitial screening tool, the present invention offers the advantages ofbeing non-invasive, and much less expensive than conventionalquantitative analyses. For these reasons, a screening test patch asdisclosed herein is particularly suited for initial screening of largepopulations such as parolees, inmates, military personnel or otherswhere monitoring is desired.

The analyte determination zone 60 and analyte reference zone 61 may bephysically separated on the patch, such as in concentric circles ordiscrete zones, as illustrated in FIG. 7, or in the case of only two orthree analytes, interspersed throughout. In the latter case, positiveresults of different determinations would be indicated by the appearanceof different colors.

A variety of well known immunoassay schemes for visualizing the presenceof an analyte of interest are well known in the art, and need not bedetailed here. However, the optimal immunoassay scheme is generally onewhich is simple and requires the fewest steps. For many types of assays,it will be desirable for the wearer to obtain rapid results such as acolor change to demonstrate a positive or negative result with as fewsteps as possible. On the other hand, drug of abuse screens are morelikely to be evaluated by clinical staff instead of by the test subject,and there is less concern for a "user friendly" product.

For example, in a concentration patch of the present invention designedfor determining both the presence of CK-MM and CK-MB enzyme, theimmobilized specific binding partner for each of those enzymes will besegregated to separate regions of the test patch. In this manner, if anenzyme-linked immunoassay system is utilized, a common enzyme and acommon substrate could be used. Alternatively, a different color is usedto express the presence of different analytes.

Another embodiment of the present invention which will indicate whethera wearer has removed the patch during the examination period isillustrated in FIG. 8. In this embodiment, the concentration patch 62 issecured to the skin 64 with an adhesive member 65. The adhesive member65 is preferably constructed of a material that is strong enough to holdthe concentration patch 62 to the skin 64, but that is relatively easilytorn such as during removal of the patch from the skin. A suitablematerial for use in this preferred embodiment is Tegaderm 1625,manufactured by Minnesota, Mining, and Manufacturing Corp. of St. Paul,Minn. Other companies, including Avery and Johnson & Johnson,manufacture similar suitable materials; the Johnson & Johnson productbeing sold under the trademark "Bioclusive." It has been found, however,that with sufficient patience, a wearer could remove an adhesive memberof this type and replace it without leaving any visible indication thatthe adhesive member has been removed. Therefore, in the particularlypreferred embodiment shown, the adhesive member 65 has stress razors 66in the form of a plurality of radial slits around its outer perimeter.The stress razors 66 can be arranged in any of a wide variety ofconfigurations and densities and accrue the advantage of tearing uponremoval, as will be apparent to one of skill in the art.

In the preferred embodiment illustrated in FIG. 8, the radial slits 66extend approximately 0.05 inches in length from the outer edge towardthe center of the concentration patch 62. The slits 66 may be arrangedwith any of a variety of regular or irregular spacings therebetween,and, in the preferred embodiment are preferably spaced approximatelyevery 0.10 inches around the perimeter of the concentration patch 62.The adhesive force of the material of the adhesive member 65 ispreferably more than the force needed to tear the adhesive member at thestress razors 66, so that if the concentration patch 62 is removed, thematerial of the adhesive member is torn. Thus, when a concentrationpatch of this preferred embodiment is worn, a torn adhesive memberserves as an indication that the wearer has likely tampered with thepatch. Of course, the weakening of the adhesive member 65 may beaccomplished by providing perforations rather than slits and the slitsor perforations may be oriented in directions other than radially.

During storage prior to use, it is desirable to cover the adhesivemember to prevent it from sticking to any surface; otherwise the stressrazors 66 could become torn prior to use. Accordingly, in the preferredembodiment shown in FIG. 8, the concentration patch is provided with aninner cover 69 to protect the adhesive member 65. The inner cover 69 isremoved to expose the adhesive member 65 prior to application of thepatch 62 to a subject's skin. Any of a variety of non-adherent materialsknow to those of skill in the art may be used for the inner cover 69,such as those commonly used to cover adhesive bandages.

The concentration patch 62 is virtually impossible to remove and replacewithout showing visible signs of tampering. Thus, any analytes in sweatproduced from skin under the concentration zone 14 during the time thepatch is worn should be present in the patch.

However, a particularly shrewd subject desiring to produce falsenegative results could obtain additional test patches. This shrewdsubject would obtain false negative results by removing the initiallyapplied test patch and replacing the test patch just prior to the timethe patch is to be removed for assay. In order to ensure that the patchremoved from the subject is the same patch which was initially appliedto the subject, an identifying marker which is difficult to reproducecan be incorporated into the patch. For example, a bar codeidentification strip 67, similar to the bar codes used at supermarketcheck out stands can be incorporated into the patch, preferably justbelow the adhesive member 65. For best results in protecting againstreplacement of the patch, it is important that the identifying markernot be easily removed and replaced without providing an indication thatthe patch has been tampered with.

In a preferred embodiment, the patch 62 has a filter 68 between theouter layer 65 and concentration zone 14, as described above inconnection with FIGS. 1-3a. In a particularly preferred embodiment, thefilter is a liquid permeable filter formed from a James River PaperDrape.

The preferred adhesive members of the embodiment shown in FIG. 8, madefrom adhesive materials, such as Tegaderm, which are relatively weak instrength, have generally been designed for hospital patients who are notexpected to perspire at high rates. Therefore, the moisture vaportransmission rate (MVTR) of these materials is relatively low. Forexample, the MVTR of Tegaderm is approximately 810 g/m*m*day. However,an active person may perspire at instantaneous rates as high as 26000g/m*m*day. Consequently, an active person may put out more sweat thanthese adhesive members can transmit to the atmosphere. If this sweataccumulates for any significant period of time, channels may be formedbetween the skin 64 and the adhesive member 65, allowing sweat to exitbetween the adhesive member and the skin, rather than be absorbed by theconcentration patch 62.

Thus, in accordance with a further embodiment of the present invention,illustrated in FIG. 9, there is provided a patch 70 having an adhesivemember 72 which allows excessive sweat to be freely transmitted to theoutside through pinhole perforations 73. The pinhole perforations may bedistributed throughout a wide band 75 extending from the outer perimeterof the adhesive member to a narrow band 77 surrounding the test region821 of the patch 70.

Sweat produced beneath test region 81, over which there are no pinholeperforations 73, will be absorbed by the test region and will not betransmitted to the outside. The test region 81 includes the area of thepatch 70 directly under the concentration zone 14 of the patch as wellas the area immediately outside this zone. The narrow band 77 outsidethe concentration zone 14 of the patch has no pinhole perforations 73,and substantially restricts sweat forming underneath the test region 81from communicating with the wide band 75 where sweat is transmitted tothe outside.

The width of the narrow band 77, is preferably between 0.025 and 0.250inches, more preferably between 0.05 and 0.125 inches. Narrow bandwidths less than the preferred width are not expected to keep contactwith the skin, whereas narrow band widths greater than the preferredwidth may allow sweat channels to form, creating a path for sweatforming within the test region 81 to communicate with the outside.

A wearer of the patch in screenings for drugs of abuse would be expectedto be rather creative in circumventing the protections of the patch. Forexample, a creative wearer could try to wash out the concentrated sweatcomponents from the patch while the patch remains on the wearer's skin.Such washing could be attempted using a needle and syringe, such asthose commonly used by intravenous drug abusers for drug injection.

For those patches employing specific binding chemistry, attemptedelution of the concentrated components using water would likely proveunsuccessful. Even for those patches not employing specific bindingchemistry for the analyte being tested, elution with water alone wouldbe difficult, requiring substantial volumes of water without triggeringthe detection of tampering through the removal of the patch from theskin. However, elution with urine from an animal or non drug user couldbe successfully used to remove certain analytes from the patch.Additionally, certain analytes could successfully be at least partiallyeluted using non-aqueous solvents such as acetone, commonly available asnail polish remover.

Thus, in order to detect tampering with the patch through elution of thepatch's contents using water or other solvents, a known amount of amarker which is readily soluble in either aqueous or non-aqueoussolvents, can be added to the concentration zone during manufacture ofthe patch. The marker should be easily quantifiable. The marker shouldalso be soluble in either aqueous or non-aqueous solvents depending onthe likely route of elution of the analyte. Additionally, the markershould be suitable for prolonged skin contact and not be readilyabsorbed by the skin. A variety of dyes used in the production of makeuphave these suitable characteristics. Oil red N (catalogue number29,849-2) sold by Aldrich Chemical Corp. of Milwaukee, Wis. is asuitable lipid soluble dye. DG01 red and DH60 yellow, both availablefrom Virginia Dare Extract Co. of Brooklyn, N.Y. are suitable watersoluble dyes. These water soluble dyes can be easily quantitated byelution from the patch followed by measuring optical density at 6500 nmfor the red or 5800 nm for the yellow dye. The quantity of dye remainingcan be compared with the range of the amount of dye found to beremaining in patches worn continuously without tampering for the samelength of time.

Non-visible markers could also be used to prevent the wearer of thepatch from obtaining feedback regarding the extent of marker remainingin the patch. A colorless protein could be used for this purpose. Aprotein should be chosen that is easily identified in the lab, and alsonot be expected in human sweat. For example, Bovine gamma globulins,such as those sold by Sigma Chemical Co. of St. Louis, Mo., could alsobe used as a marker. The presence of these markers can be easilyascertained using Bovine IgG RID kit, available from ICN of Coasta Mesa,Calif.

Thus, when a suitable marker is employed within the concentration patch,when the patch is analyzed for the particular analyte being tested, thepatch can also be analyzed for the presence of the marker. For visiblemarkers, such as makeup dyes, the presence of the marker may be analyzedby simply viewing the patch. For non-visible markers, the non-visiblemarker can be assayed along with the analyte. A significant decrease inthe amount of marker present would be an indication of tampering throughelution of the patch with a solvent.

A further method of tampering with the patch would be to add anadulterant to the patch which interferes with the assay chemistry.Numerous materials have been used to adulterate urine tests for drugs ofabuse. The most commonly used, and generally most effective method ofproducing a false negative result in a urine test is to dilute the urineby ingestion of excessive amounts of fluids. Advantageously, thisapproach would not likely be successful in producing flase negativeresults in the sweat collection patch of the present invention becauseinterstial concentration of drug metabolites is less likely to beinfluenced by ingestion of fluids.

However, the addition of certain adulterants to the patch may interferewith the analysis chemistry. For example, acids and bases are known tointerfere with assays for many drug metabolites by altering themetabolites' molecular structure. Additionally, many household products,such as detergents, ammonia, ascorbic acid (Vitamin C), and drainopeners have been used to interfere with urine assays. These productsall produce extremes of pH and would be expected to result in trauma tothe skin if used in connection with tests using the concentration patchof the present invention. This trauma could be noted by the technicianremoving the patch.

However, weak acids and bases, as well as eye drops sold under thetrademark "Visine," are also known to interfere with a variety of assaysfor drug metabolites in urinalysis. However, these materials would notbe expected to produce skin trauma. Thus, the use of these materials orother compounds interfering with an assay that do not cause skin traumamight go unnoticed by the technician removing the patch if the liquidcontents of the material have had time to evaporate across the outerlayer of the patch. However, "Visine" and most other adulterants wouldbe expected to contain ionic materials.

Thus, in order to detect the use of an adulterant, test strips can beincorporated into the concentration patch which will detect the presenceof various ionic materials or of extremes of pH. Litmus paper, such asHydrion pH test paper, available from Baxter Scientific Products, iswell known as an indicator of variances of pH. Accordingly, a shortpiece, for example 1 cm by 1/2 cm, of litmus paper could be incorporatedinto the patch to detect the various household products identified abovewhich are known to be highly acidic or basic.

Many test strips are also known for detecting the presence of ionicmaterials. For example Baxter Scientific Products supplies test stripsfrom a variety of manufacturers for the detection of each of thefollowing ions: aluminum, ammonium, chromate, cobalt, copper, ion,nickel, nitrate, peroxide, sulphite, tin, and calcium. In addition, teststrips sold under the name "Qantab" are available from Baxter ScientificProducts which identify the presence of chlorine ions. Other test stripsavailable from the same supplier show glucose, protein, and ketones.Most of these test strips are read by simply comparing the color of thestrips with a color chart included with the strips. Thus, the teststrips provide a simple method of identifying the introduction of any ofa variety of adulterant materials.

In order to detect adulterants, such as "Visine," which contain ionicmaterials not known to the person performing the test, the tester mustfirst assay the adulterant using a variety of test strips for ions toascertain which ions are present in the materials. Once the appropriateions are detected, the test strips corresponding to those ions can beincorporated into the concentration patch in order to provide anindication that the adulterant has been added to the patch.

Curiously, any particular adulterant might produce false negativeresults in some assays and false positive results in others. For eachassay, the common adulterants which could be used to produce falsenegative results could be identified by testing the assays with theaddition of small amounts of these known materials. Test strips couldthen be included which would detect the addition of these adulterants.

In a preferred embodiment, the test strip or strips are placed facingthe skin, where the strips are not visible to the wearer. The wearer isthereby not provided any feedback which aids the wearer in deception.

Many biological compounds are known to be affected by various spectralbands of light energy. For example, urine samples for analysis of LSDmust be kept from exposure to strong light. (Schwartz, Arch. Inter. Med.148:2407-12 (1988)). Further examples of compounds which requireprotection from light include cocaine hydrochloride (Martindale ExtraPharmacopoeia, 29th Ed., p. 1213) and morphine sulphate (Id., p. 1310).It is expected that these and other compounds may be affected byexposure to light while being concentrated in the collection patch aswell.

Many analytes to be determined by a concentration patch of the presentinvention may require collection and storage in the patch for prolongedperiods of time (up to several weeks). These analytes are, therefore,exposed to substantial quantities of photoradiation. This quantity ofphotoradiation may be substantially greater than during a urine assayfor the same or similar analyte. Also, many analytes have peculiarlyhigh sensitivity to light. Thus, for analytes of peculiarly highphotosensitivity or for those requiring prolonged collection andstorage, it is particularly important to shield photosensitive analytesfrom light during prolonged storage in the patch.

Accordingly, in still another embodiment of the present invention,illustrated in FIG. 10, there is provided a test patch 90 having a lightattenuation layer 92 between the outer adhesive layer 65 and theconcentration zone 14. In FIG. 10, the adhesive layer 65, is shownhaving stress razors 66, however, this feature is to be understood asbeing optional in this embodiment of the invention.

The attenuation layer 92 is provided in order to attenuate thetransmission of light into the concentration zone 14 where thebiological compound of interest is being collected and stored. The layer92 should be substantially impervious to the transmission ofphotoradiation, yet should also allow relatively unrestricted passage ofthe aqueous components of sweat to the outer adhesive layer 65. Thelayer 92 should be of sufficient porosity that diffusion of the aqueouscomponents of sweat occurs at least as rapidly as sweat normallyaccumulates in the patch.

Because light of may wavelengths is capable of degrading the variousbiological compounds which may be of interest, the layer 92 should haveoptical properties which attenuate light throughout a wide spectrum.Attenuation can be achieved by either reflection or absorption ofincoming light. Reflection may be achieved through, for example, the useof any of a variety of metallic surfaces. When used in accordance withcertain preferred embodiments of the present invention, the attenuationlayer 92 should allow passage of aqueous components of sweat. In orderto provide a reflective layer with the suitable permeability, thinmetallic foil with small holes can be provided. For example, aluminumfoil, commercially available from many sources including ReynoldsAluminum Co., could be perforated with a plurality of small holes.

Absorptive attenuation layers can be provided through the use of a blacksurface. Preferably, these surfaces would continue to allow permeabilityof aqueous components of sweat. It is important that any dye orpigmentation in the attenuation layer 92 not bleed when exposed to theaqueous components of sweat and also that it not interfere with anybinding chemistry or in the analysis of the analyte. Any of a variety ofthin black papers having these properties are commercially available andare suitable for use as in the attenuation layer. For example, blackDeltaware cellulose membrane filters available from Baxter ScientificProducts have been found to be especially useful for use as anattenuation layer. This product is available in a variety of porosities;more open pores are preferred. Thus, in the preferred embodiment, 0.6micron black Deltaware filters are provided.

In an alternative to the provision of an attenuation layer (not shown),the adhesive layer 65 can be made to attenuate light, either throughabsorption or reflection. As an example of an absorptive adhesive layer,black colorant, such as fine carbon black powder, could be incorporatedinto the extrusion of the adhesive sheet.

The following examples describe only specific applications of thepresent invention.

EXAMPLE 1 Preparation of Microbead Test Patch

One specific application of the present invention is the dualdetermination of skeletal muscle and cardiac muscle status as a resultof exercise. A dermal concentration patch is constructed in accordancewith the embodiment illustrated at FIGS. 3 and 3a. The gauze layer isprepared by cutting a circular patch having an approximately 1-inchdiameter from a Johnson & Johnson non-stick gauze pad. The inner andouter porous layers are next prepared by cutting two circular patches ofUltipor (nylon 6), from Pall Corporation in Glen Cove, N.Y. Ultipormembrane is both liquid permeable and microporous, and a membrane isselected having, for example, a 1 micron rating. The microbead layer isprepared by covalently bonding monoclonal antibody raised against CK-MBto a multiplicity of polystyrene beads having a mean particle size of atleast about 10 microns.

The concentration patch is assembled by distributing approximately 0.2gram of microbeads across the surface of one of the porous layers. Thesecond porous layer is thereafter disposed adjacent the microbeads, andthe gauze layer is next placed on top of the second porous layer. Atthis point, the patch is upside-down. The peripheral edges of each ofthe first and second porous layers and the gauze layers are securedtogether by conventional heat-sealing techniques. Thereafter, thesubassembly is turned over and an annular torus of adhesive tape havingapproximately a 2-inch outside diameter and slightly less than a 1-inchinside diameter is secured thereto to produce a finished concentrationpatch.

EXAMPLE 2 Cardiac Muscle Status Test

The concentration patch of Example 1 is then secured to the chest of ahealthy 40-year old male and worn throughout a 36-mile (130-minute)bicycle ride. Upon removal of the concentration patch following theride, the test patch is immersed in a first solution containing anexcess of enzyme labeled anti-CK-MB for approximately 30 minutes, topermit conjugation of labeled antibody with immobilized analyte. Thepatch is then rinsed under tap water to remove unbound labeled antibodyand immersed in a second solution containing a substrate for the boundenzyme label, which undergoes a color change when acted upon by theenzyme. Appearance of color through the top porous layer indicates thepresence of CK-MB, and possible cardiac injury. Comparison to a colorchart permits rough quantification.

EXAMPLE 3 Test for Use of Marijuana

THC polyclonal antibody from sheep (available from Biogenesis,Bournmouth, England) is diluted 1:100 in PBS (pH 7.5). The antibodiesare bound to Gelman 0.45μ (SU-450) Ultrabind Supported Membrane,following the protocol in Gelman Original Equipment Manufacturerapplication P.N. 31,084. The membranes are air dried. Disks, 3/8 inch indiameter, are cut from the coated Gelman membranes. These 3/8 inch disksare mounted at the center of a 1/4 inch diameter hole cut in the centerof a one inch diameter circle of Tegaderm 1625 Transparent Dressing(available from Minnesota Mining and Manufacturing, St. Paul, Minn.).

Three mounted membranes are secured to the chest of a subject who thensmokes a marijuana cigarette. Three mounted membranes are also securedto a subject who has never used marijuana in any form and who agrees notto use it for the next seven days. The membranes remain in place untilthey are removed, seven days later. Each of the removed membranes isflushed five times with 300 μl of 0.2% Tween 20 in PBS. The membranesare incubated for 30 minutes in 100 μl of E-Z Screen Cannabinoid enzymeconjugate from the E-Z Screen Test Kit (available from EnvironmentalDiagnostics, Inc., Burlington, N.C.).

After incubation, each membrane is flushed three times with 300 μl of0.2% Tween 20 in PBS, followed by three flushes with PBS alone. Themembranes are then incubated in TMB Membrane Peroxide Substrate(available from Kirkegaard & Perry Labs, Gaithersburg, Md.) for 10minutes. A light blue background appears in all six membranes. Whitedots appear over the background on the three membranes taken from thesubject who smoked a marijuana cigarette, indicating sweat gland outputof sweat containing THC derivatives. No white dots appear on the threemembranes taken from the subject who has never used marijuana.

EXAMPLE 4 Positive Control Patch

Mouse anti-human IgG, Fc monoclonal antibody (available from ICN, CostaMesa, Calif.) is diluted 1:100 in PBS (pH 7.5). The antibodies are boundto Gelman 0.45μ (SU-450) Ultrabind Supported Membrane, following theprotocol in Gelman Original Equipment Manufacturer application P.N.31,084. The membranes are air dried. Disks, 3/8 inch in diameter, arecut from the coated Gelman membranes. These 3/8 inch disks are centeredand mounted on a 1/4 inch diameter hole cut in the center of a one inchdiameter circle of Tegaderm 1625 Transparent Dressing.

Three mounted membranes are secured to the chest of five human subjects.The membranes remain in place until they are removed, seven days later.Each of the removed membranes is flushed five times with 300 μl of 0.2%Tween 20 in PBS. The membranes are incubated for 30 minutes in 100 μl ofHorseradish peroxidase enzyme conjugated to goat anti-human IgG, Fcpolyclonal antibody (available from ICN, Costa Mesa, Calif.) diluted1:1000 in PBS.

After incubation, each membrane is flushed three times with 300 μl of0.2% Tween 20 in PBS, followed by three flushes with PBS alone. Themembranes are then incubated in TMB Membrane Peroxide Substrate(available from Kirkegaard & Perry Labs, Gaithersburg, Md.) for 10minutes. Blue dots corresponding to individual sweat ducts appear overthe background on all of the membranes, indicating that the chemistry ofthe patches is operative by their detection of the IgG expected in thesweat of all subjects.

Although this invention has been described in terms of certain preferredembodiments and immunoassay schemes, other embodiments and immunoassaysthat are apparent to those of ordinary skill in the art are also withinthe scope of this invention. Accordingly, the scope of the invention isintended to be defined only by reference to the appended claims.

We claim:
 1. A dermal concentration patch for determining the presenceof an analyte in a subject mammal's perspiration, comprising:a waterpermeable support layer having a first and a second side; at least onefirst and at least one second reagent immobilized in the support layer;and adhesive tape, said adhesive tape having an upper and lower surface,said lower surface of said adhesive tape facing said second side of saidsupport layer, said lower surface of said adhesive tape furthercomprising an adhesive substance and being adapted to secure the firstside of the support layer in fluid communication with the subjectmammal's skin, said adhesive tape comprising a plurality of stressrazors disposed thereon so that the tape cannot readily be removed as aunitary whole, wherein water is permitted to escape through the supportlayer to the outside of the concentration patch, wherein the firstreagent comprises a specific binding partner for the analyte to bedetermined, and wherein the second reagent comprises a specific bindingpartner for a reference substituent in the perspiration.
 2. A method ofdetecting false negative results in an assay of a body fluid from asubject, which false negative results are the result of noncompliancewith a testing procedure of said assay by the subject, comprising thesteps of:securing a test patch to a subject with adhesive tape, saidadhesive tape having a plurality of stress razors disposed thereon, saidtest patch being secured such that it is placed in communication with asource of body fluid; allowing the test patch to remain in place for asufficient test period to allow the presence of analyte to bedetermined; and thereafter examining the test patch to determine whetherthe test patch is torn, a torn test patch indicating that the test patchmay not have been continuously secured to the subject.
 3. A dermalconcentration patch for determining the presence of an analyte in asubject mammal's perspiration, in which false negative results producedthrough the subject's removal of the patch and replacement with anotherpatch can be detected, said patch comprising:a water permeable supportlayer having a first side and a second side, said first side beingcapable of being placed in fluid communication with a subject's skin;means for removably securing the first side of the support layer influid communication with the subject's skin; and an identifying markerincorporated into the patch which is unique to the patch and which isdifficult for the subject to reproduce, the marker thereby identifyingthe patch as the patch which was initially applied to the subject,wherein the absence of the unique identifying marker in the patch afterthe patch is removed is indicative of false results.
 4. The dermalconcentration patch of claim 3, wherein the identifying marker is a barcode disposed in a position hidden from view of the subject.
 5. Thedermal concentration patch of claim 4, wherein the identifying marker isdisposed between the support layer and the means for removably securingthe patch, thereby disposing the marker in a position hidden from viewof the subject.
 6. A dermal concentration patch for determining thepresence of a drug of abuse or metabolite thereof in a subject mammal'sperspiration, said patch being wearable by the subject for a test periodof time, comprising:a water permeable support layer in which said drugof abuse or metabolite thereof can be concentrated; means for removablysecuring the support layer in fluid communication with the subject'sskin, wherein said means is water permeable; and a false test resultdetector within said patch, said false test result detector providing anindication at the end of said test period of time if said patch has beentampered with, wherein water is permitted to escape through said supportlayer to the outside of said concentration patch, thereby allowing saiddrug of abuse or metabolite thereof to be concentrated in said supportlayer.
 7. A dermal concentration patch according to claim 6, wherein thefalse test result detector comprises a reagent which is a specificbinding partner for a reference substituent in perspiration.
 8. A dermalconcentration patch according to claim 7, wherein the reagent isimmobilized in the support layer.
 9. A dermal concentration patchaccording to claim 6, wherein the false test result detector comprises asoluble marker within the patch, the presence of said marker beingdetectable after completion of the test period, wherein a substantialloss in the quantity of soluble marker present in the patch isindicative of a false negative result.
 10. A dermal concentration patchfor determining the presence of a drug of abuse or metabolite thereof ina subject mammal's perspiration, said patch being wearable by thesubject for a test period of time, comprising:a water permeable supportlayer in which said drug of abuse or metabolite thereof can beconcentrated; means for removably securing the support layer in fluidcommunication with the subject's skin; and a false test result detectorwithin said patch, said false test result detector providing anindication at the end of said test period of time if said patch has beentampered with, wherein the means for removably securing comprises anadhesive layer and wherein the false test result detector comprises aplurality of stress razors disposed on the adhesive layer so that theadhesive layer cannot readily be removed as a unitary whole.
 11. Adermal concentration patch for determining the presence of a drug ofabuse or metabolite thereof in a subject mammal's perspiration, saidpatch being wearable by the subject for a test period of time,comprising:a water permeable support layer in which said drug of abuseor metabolite thereof can be concentrated; means for removably securingthe support layer in fluid communication with the subject's skin; and afalse test result detector within said patch, said false test resultdetector providing an indication at the end of said test period of timeif said patch has been tampered with, wherein the false test resultdetector comprises an identifying marker unique to said patch which isdifficult for the subject to reproduce, the marker thereby identifyingthe patch as the patch which was initially applied to the subject,wherein the absence of the unique identifying marker in the patch afterthe patch is removed is indicative of false negative results.
 12. Adermal concentration patch according to claim 11, wherein theidentifying marker is a bar code disposed in a position hidden from viewof the subject when the patch is worn by the subject.
 13. A dermalconcentration patch according to claim 12, wherein the identifyingmarker is disposed between the support layer and the means for removablysecuring the support layer, thereby disposing the marker in a positionhidden from the view of the subject when the patch is worn by thesubject.
 14. A method of determining the presence of a drug of abuse ormetabolite thereof in a subject mammal's perspiration with a dermalconcentration patch, comprising:removably securing a dermalconcentration patch comprising a water permeable support layer in fluidcommunication with the subject's skin; accumulating perspiration fromsaid subject in said support layer for a test period of time; permittingwater to escape through the support layer to the outside of said patch,thereby concentrating the drug of abuse or metabolite thereof in thesupport layer; and detecting false test results, if present.
 15. Themethod of claim 14, wherein the detecting step comprises detectingwhether the patch has been worn by the subject for substantially all ofthe test period.
 16. The method of claim 15, wherein the detecting stepcomprises binding a reference substituent in perspiration with a reagentin said support layer which is a specific binding partner of saidsubstituent.
 17. A method of determining the presence of a drug of abuseor metabolite thereof in a subject mammal's perspiration,comprising:removably securing a water permeable support layer in fluidcommunication with the subject'skin; accumulating perspiration from saidsubject in said support layer for a test period of time; permittingwater to escape through the support layer, thereby concentrating thedrug of abuse or metabolite thereof in the support layer; and detectingfalse test results if present; wherein the securing step comprisessecuring said support layer to the subject using adhesive materialcomprising a plurality of stress razors disposed on said material suchthat the material cannot be readily removed as a unitary whole.
 18. Themethod of claim 17, wherein the detecting step comprises detectingwhether the adhesive material has been torn.
 19. A method of determiningthe presence of a drug of abuse or metabolite thereof in a subjectmammal's perspiration, comprising:removably securing a water permeablesupport layer in fluid communication with the subject's skin;accumulating perspiration from said subject in said support layer for atest period of time; permitting water to escape through the supportlayer, thereby concentrating the drug of abuse or metabolite thereof inthe support layer; and detecting false test results if present; whereinthe securing step comprises securing the support layer in combinationwith a concentration patch, and wherein the detecting step comprisesincluding in said concentration patch an identifying marker unique tosaid patch which is difficult for the subject to reproduce and detectingwhether the unique identifying marker identifying the patch secured tothe subject is absent in the patch which is removed from the patient atthe end of the test period.
 20. The method of claim 19, wherein saididentifying marker comprises a bar code disposed in a position hiddenfrom view of the subject when the patch is worn by the subject, saiddetecting step further comprising detecting the presence or absence ofsaid bar code.
 21. A method of detecting false negative results in anassay of a body fluid from a subject, which false negative results arethe result of noncompliance with a testing procedure of said assay bythe subject, comprising the steps of:securing a test patch to a subjectwith adhesive tape, said test patch being secured such that it is placedin communication with a source of body fluid, said test patch havingincorporated therein an identifying marker unique to said patch which isdifficult for the subject to reproduce, the marker thereby identifyingthe patch as the patch which was initially applied to the subject;allowing the test patch to remain in place for a sufficient test periodto allow the presence of analyte to be determined; and thereafterexamining the test patch to detect whether the unique identifying markeridentifying the patch secured to the subject is absent in the patchwhich is removed from the patient at the end of the test period, whereinthe absence of the unique identifying marker in the patch when the patchis examined is indicative of false negative results.
 22. The method ofclaim 21, wherein said identifying marker comprises a bar code disposedin a position hidden from view of the subject when the patch is worn bythe subject.
 23. A dermal concentration patch for determining thepresence of a drug of abuse or metabolite thereof in a subject mammal'sperspiration, said patch being wearable by the subject for a test periodof time, comprising:a water permeable support layer in which said drugof abuse or metabolite thereof can be concentrated, said support layerhaving a first and second side; adhesive tape for removably securing thesupport layer in fluid communication with the subject's skin, saidadhesive tape having an upper and lower surface, said lower surface ofsaid adhesive tape facing said second side of said support layer, saidlower surface of said adhesive tape further comprising an adhesivesubstance and being adapted to secure the first side of the supportlayer in fluid communication with the subject mammal's skin; and a falsetest result detector within said patch, said false test result detectorproviding an indication at the end of said test period of time if saidpatch has been tampered with, wherein the false test result detectorcomprises a chemical marker not readily absorbed by said subjectmammal's skin, and wherein a decrease in the amount of said marker insaid patch is indicative of false negative results.
 24. A method ofdetermining the presence of a drug of abuse or metabolite thereof in asubject mammal's perspiration, comprising:removably securing a waterpermeable support layer in fluid communication with the subject's skin;accumulating perspiration from said subject in said support layer for atest period of time; and detecting false test results, if present;wherein the securing step comprises securing the support layer incombination with a concentration patch, wherein the detecting stepcomprises detecting in said concentration patch a chemical marker notreadily absorbed by said subject mammal's skin, and wherein a decreasein the amount of said marker which is detected in said patch isindicative of false negative results.